There is still much unknown about how nitric oxide (NO) biosynthesis by NO synthase (NOS) is tightly regulated at the molecular level. This is remarkable because deviated NO production in vivo has been implicated in an increasing number of serious diseases lacking effective treatments, including stroke, septic shock and cancer. Unlike inducible NOS, endothelial and neuronal NOS isoforms (eNOS and nNOS) are controlled by calmodulin (CaM) through facilitating catalytically significant interdomain electron transfer (IET) processes. It is proposed that CaM activates NO synthesis in eNOS and nNOS through a conformational change of the flavin mononucleotide (FMN) domain from its shielded electron-accepting (input) state to a new electron-donating (output) state. The FMN-heme IET within the NOS output state is essential for NO synthesis at the catalytic heme. However, the mechanism for formation of the NOS output state remains unclear, and this stands as a critical barrier for understanding regulation of NOS catalysis at the molecular level. The focus of this study is to investigate the molecular mechanism of CaM-activated output state formation in full length human eNOS and nNOS enzymes. We hypothesize that productive FMN/heme interactions, specific binding of CaM to NOS, and unique autoinhibitory insert in the FMN domain synergistically control formation of the output state for NO production. This hypothesis will be tested by quantitating kinetics of the discrete FMN-heme IET step in the enzymes through three complementary and synergistic Aims. We have developed innovative laser flash photolysis approaches to determine the FMN-heme IET kinetics as a direct measure of formation of the NOS output state. The experimental design will integrate our laser flash photolysis methodology and state-of-art pulsed electron paramagnetic resonance (EPR) techniques with site-directed mutagenesis. This study will significantly improve the fundamental understanding of NOS regulation at the molecular level, and will provide new important insight as to how NOS might be selectively modulated for therapeutic purposes. The mechanism for formation of the NOS output state has long been understudied due to lack of reliable techniques for determining the FMN-heme IET kinetics. Our innovative laser flash photolysis methodology sets the stage for a detailed investigation of NOS regulation through formation of the output state. Given the novel and exploratory nature of this experimental venture, this study is well suited for an R21 award.